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International audience; The adult non-obese Goto–Kakizaki (GK) rat model of type 2 diabetes, particularly females, carries in addition to hyperglycaemia a genetic predisposition towards dyslipidaemia, including hypercholesterolaemia. As cholesterol-induced atherosclerosis may be programmed in utero, we looked for signs of perinatal lipid alterations and islet microangiopathy. We hypothesise that such alterations contribute towards defective pancreas/islet vascularisation that might, in turn, lead to decreased beta cell mass. Accordingly, we also evaluated islet inflammation and endothelial activation in both prediabetic and diabetic animals. Blood, liver and pancreas were collected from embryonic day (E)21 fetuses, 7-day-old prediabetic neonates and 2.5-month-old diabetic GK rats and Wistar controls for analysis/quantification of: (1) systemic variables, particularly lipids; (2) cholesterol-linked hepatic enzyme mRNA expression and/or activity; (3) pancreas (fetuses) or collagenase-isolated islet (neonates/adults) gene expression using Oligo GEArray microarrays targeted at rat endothelium, cardiovascular disease biomarkers and angiogenesis, and/or RT-PCR; and (4) pancreas endothelial immunochemistry: nestin (fetuses) or von Willebrand factor (neonates). Systemic and hepatic cholesterol anomalies already exist in GK fetuses and neonates. Hyperglycaemic GK fetuses exhibit a similar percentage decrease in total pancreas and islet vascularisation and beta cell mass. Normoglycaemic GK neonates show systemic inflammation, signs of islet pre-microangiopathy, disturbed angiogenesis, collapsed vascularisation and altered pancreas development. Concomitantly, GK neonates exhibit elevated defence mechanisms. These data suggest an autoinflammatory disease, triggered by in utero programming of cholesterol-induced islet microangiopathy interacting with chronic hyperglycaemia in GK rats. During the perinatal period, GK rats show also a marked deficient islet vascularisation in conjunction with decreased beta cell mass.

1. A recently recognized complication of uncontrolled diabetes mellitus is the encephalopathy involving, among other regions, the hippocampus. Since estrogens bring neuroprotection in cases of brain injury and degenerative diseases, we have studied if estradiol (E2) administration counteracts some hippocampal abnormalities of streptozotocin (STZ)-diabetic adult mice. 2. We first report the ability of E2 to modulate neurogenesis in the dentate gyrus (DG) and subventricular zone (SVZ) of diabetic mice. Using bromodeoxyuridine (BrdU) to label newly generated cells, a strong reduction in cell proliferation was obtained in DG and SVZ of mice sacrificed 20 days after STZ administration. The reduction was completely relieved by 10 days of E2 pellet implantation, which increased 30-fold the circulating E2 levels. 3. Diabetic mice also showed abnormal expression of astrocyte markers in hippocampus. Thus, increased number of GFAP(+) cells, indicative of astrogliosis, and increased number of apolipoprotein-E (Apo-E)(+) astrocytes, a marker of ongoing neuronal dysfunction, was found in stratum radiatum below the CA1 hippocampal subfield of diabetic mice. Both parameters were reverted to normal by the E2 regime that upregulated cell proliferation. 4. The studies demonstrated that hippocampal neuropathology of uncontrolled diabetes is a reversible condition and sensitive to estrogen treatment. Studies in animal models may open up new venues for understanding the beneficial role of steroid hormones in diabetic encephalopathy.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which insulin-producing beta cells of the pancreatic islets of Langerhans are destroyed. The nonobese diabetic (NOD) mouse is one of the rare spontaneous models that enable the study of prediabetic pancreatic events. The etiology of the autoimmune attack in human and animal T1D is still unknown, but genetic and environmental factors are involved in both cases. Although several autoantigens have been identified and defective immune-system regulation is implicated, this information does not satisfactorily explain the generally accepted beta-cell specificity of the disease or how so many and diverse environmental factors intervene in its pathogenesis. Based on data obtained from evaluating glucose homeostasis in a variety of situations, particularly stress and cytokine administration, in young prediabetic NOD mice, the author hypothesizes that the islet of Langerhans is a major actor, and its altered regulation through environmentally induced insulin resistance might reveal latent T1D. It is also postulated that T1D pathogenesis might be linked to abnormal pancreas development, probably due to disturbances of glutamic acid decarboxylase (GAD) + innervation phagocytosis by defective macrophages during the early postnatal period. Also discussed is the role of defective presentation of pancreatic hormones and GAD in the thymus, and its potential repercussion on T-cell tolerance. Observations have demonstrated that the diabetogenic process in the NOD mouse is extremely complex, involving neuroendocrine immune interaction from fetal life onward.

The mouse pancreas, an immature organ at birth, reaches its adult size and morphology after weaning (3 weeks of age). Around this time, apoptotic phenomena and various types of macrophages are normally present. During development, Fas-Fas ligand (FasL) interactions are known to play a role in apoptotic events involved in tissue remodeling and elimination of damaged cells, and macrophages are routinely observed near apoptotic cells. Apoptosis and Fas-FasL interactions are also thought to be involved in the pathogenesis of autoimmune diseases, particularly type 1 diabetes (T1D). Therefore, we used early postnatal mouse pancreata from three control strains (C57BL/6, DBA/2, BALB/c) and from two strains with the nonobese diabetic (NOD)-related genetic background (the spontaneous T1D NOD model and the lymphocyte-deficient NODscid strain) to study apoptotic phenomena together with the molecular and immunohistochemical expression of proapoptosis (Fas, FasL) and antiapoptosis (Bcl-2) proteins. First, although no major difference in the numbers of total pancreatic apoptotic cells was noted among strains, significantly more FasL(+) expression was detected immunohistochemically in mice with the NOD genetic background than in control pancreata from birth to 1 month of age. Second, FasL(+), Fas(+), and Bcl-2(+) structures seemed to be associated with innervation, regardless of the strain and age. Third, in control and NOD strains, nerves (identified by immunohistochemical labeling of peripherin or neurofilament 200), were often observed in periductular and peri-insular areas. Finally, some peripherin-positive nerves expressed the interferon-inducible protein-10 chemokine, and various types of macrophages were found to be in close proximity. These data highlight an overlooked, innervation-related aspect of normal mouse postnatal pancreas development with possible implications in T1D pathogenesis.

Glucagon-producing α cells play a crucial role during the perinatal period. Because of their peri-islet localization near the early dendritic and macrophage cell infiltration, we thought it pertinent to investigate α cells in greater depth in nonobese diabetic (NOD) mice, a well-recognized spontaneous model for human type I diabetes. We determined α-cell distribution (glucagon immunohistochemistry and image analysis) and activity (real-time reverse transcriptase polymerase chain reaction [RT-PCR] and glucagon radioimmunoassay [RIA]), in relationship to glycemia in NOD and lymphocyte-deficient NODscid mice as compared to control mice (C57BL/6) from birth onward. NOD and NODscid mice, particularly at 1 day of age, had twice as many very small islets (

The expression of preproinsulin (ppIns), proglucagon, prosomatostatin, and propancreatic polypeptide was investigated in thymic extracts, thymic cells, and thymic cell lines from C57BL/6 mice by RT-PCR. The expression of pancreatic hormones was similar in thymic extracts taken from neonatal and 2-, 4-, and 8-week-old animals, but was decreased in 20-week-old animals. Pancreatic hormone expression was not observed in mouse liver, salivary gland, or spleen. Analysis of thymic cell populations revealed a 10- to 20-fold enrichment in expression of all hormones in low buoyant density cells. No expression was detected in high buoyant density cells (predominantly thymocytes) or in thymic epithelial cell lines, primary cultures of epithelial cells, or peripheral macrophages. In addition, immunoreactive insulin, measured by specific RIA, was detectable in the low buoyant density population, but not in high buoyant density cells. The enriched cell population was depleted of contaminating lymphocytes and sorted based on reactivity to the cell surface markers F4/80 (macrophage) or N418 (dendritic cells). Cells gated for N418 demonstrated expression for ppIns, but not the other pancreatic hormones. Conversely, expression for proglucagon, prosomatostatin, and propancreatic polypeptide, but not ppIns, was detected in F4/80-gated cells. Our data indicate that pancreatic endocrine hormones are differentially expressed by dendritic cells and macrophages in a normal mice., Instituto de Investigaciones Bioquímicas de La Plata

Because few data were available on glucose homeostasis at the early prediabetic stage in the nonobese diabetic (NOD) mouse, we investigated glycemia, insulinemia, and pancreatic insulin content under basal conditions in both sexes of 4-, 6-, and 8-week-old fed NOD mice, compared with sex- and age-matched fed C57BL/6 mice. We also investigated glucose tolerance in both sexes of fasting 8-week-old NOD and C57BL/6 mice. The main results obtained under basal fed conditions, when comparing both strains, were lower glycemia and higher insulinemia in NOD females at all ages investigated and in NOD males (particularly at 6 weeks of age). Glucose tolerance tests showed that: 1) the blood glucose response to 1 g/kg i.p. glucose was less sustained in both sexes of 8-week-old NOD mice than in their control counterparts; 2) the blood insulin response to glucose (1 g/kg i.p.) appeared earlier in both sexes of NOD mice than in sex-matched C57BL/6 mice; 3) an unusual sexual dimorphism existed in NOD mice, compared with controls, with females secreting, in response to glucose, twice as much insulin as males; 4) dose-response studies (1-6 g/kg glucose) confirmed the lower increase in blood glucose levels in both sexes of NOD mice and their unusual sexual dimorphism in insulin secretion; and 5) glucose tolerance tests in 4- to 8-week-old NOD mice showed that although the sexual dimorphism in insulin secretion was not observed in 4-week-old mice, it was particularly striking at 6 weeks of age. Taken together, these results suggest that beta-cell hyperactivity exists in the NOD mouse at the early prediabetic stage, especially in NOD females.

International audience; Regenerating (REG) proteins are associated with islet development, b-cell damage, diabetes and pancreatitis. Particularly, REG-1 and REG-3-beta are involved in cell growth/survival and/or inflammation and the Reg1 promoter contains interleukin-6 (IL-6)-responsive elements. We showed by transcriptome analysis that islets of Goto-Kakizaki (GK) rats, a model of spontaneous type 2 diabetes, overexpress Reg1, 3a, 3b and 3c, vs Wistar islets. Goto-Kakizaki rat islets also exhibit increased cytokine/chemokine expression/release, particularly IL-6. Here we analyzed Reg1 and Reg3b expression and REG-1 immuno-localization in the GK rat pancreas in relationship with inflammation. Isolated pancreatic islets and acinar tissue from male adult Wistar and diabetic GK rats were used for quantitative RT-PCR analysis. REG-1 immunohistochemistry was performed on paraffin sections with a monoclonal anti-rat REG-1 antibody. Islet cytokine/chemokine release was measured after 48 h-culture. Islet macrophage-positive area was quantified on cryostat sections using anti-CD68 and major histocompatibility complex (MHC) class II antibodies. Pancreatic exocrine-to-endocrine Reg1 and Reg3b mRNA ratios were markedly increased in Wistar vs GK rats. Conversely, both genes were upregulated in isolated GK rat islets. These findings were unexpected, because Reg genes are expressed in the pancreatic acinar tissue. However, we observed REG-1 protein labeling in acinar peri-ductal tissue close to islets and around large, often disorganized, GK rat islets, which may retain acinar cells due to their irregular shape. These large islets also showed peri-islet macrophage infiltration and increased release of various cytokines/ chemokines, particularly IL-6. Thus, IL-6 might potentially trigger acinar REG-1 expression and secretion in the vicinity of large diabetic GK rat islets. This increased acinar REG-1 expression might reflect an adaptive though unsuccessful response to deleterious microenvironment.

Prophylactic insulin treatment has been shown to have beneficial effects in type 1 diabetes, both in humans and in various animal models of the disease. In experimental models, the protective effect of prophylactic insulin treatment was observed in two parameters: (1) progression of insulitis and (2) diabetes incidence. The mechanism of protection still remains to be investigated. We therefore analysed by immunohistochemistry the effect of prophylactic insulin treatment vs placebo treatment (from 4 to 13 weeks of age) on ER-MP23+ macrophage infiltration in and around pancreatic islets in the non-obese diabetic (NOD) mouse, a spontaneous model for type 1 diabetes. BALB/c mice were used as diabetes-free controls. Using conventional haematoxylin-eosin staining, we detected a protective effect of prophylactic insulin treatment in NOD females on the lymphocytic insulitis, significant at 13 weeks, but not at 9 weeks of age. However, when assessed by immunostaining for early infiltration of ER-MP23+ macrophages around islets, the reduction in severity of insulitis could already be detected as early as 9 weeks of age. With regard to the early accumulation of ER-MP23+ cells, we observed that their numbers per mm2 surface area of the exocrine pancreas and per micron at the circumference of the islet were higher in placebo-treated NODs (197 +/- 13.8 and 14 +/- 0.9, respectively) as compared to age-matched BALB/c mice (123.1 +/- 7.1 and 3.5 +/- 0.9, respectively). Prophylactic insulin treatment of NODs lowered the attraction of ER-MP23+ macrophages to the exocrine pancreas and to the circumference of the islets (156.3 +/- 8.5 and 7.9 +/- 1, respectively). Interestingly also, the islet size was found to be larger in placebo-treated NODs (51% was larger than 10 microns2) than in age-matched BALB/c mice (9% larger than 10 microns2). Prophylactic insulin treatment of NODs reduced their islet size to sizes found in the control BALB/c strain. In conclusion, the decrease in islet size by early insulin administration, and the lower attraction of ER-MP23+ macrophages to the islets are morphological indications that prevention of diabetes development by prophylactic insulin treatment results from a down-regulation of islet metabolism and growth, with a concomitant decline in the release of islet factors attracting macrophages.

Diabetes mellitus is associated with increased risk for cardiovascular disorders, which are major causes of mortality in this disease. Hyperhomocysteinemia, defined by high plasma homocysteine levels, is an independent risk factor for the development of cardiovascular diseases. Type 2 diabetic patients have higher circulating homocysteine levels than healthy subjects and these levels are even higher in plasma of obese than nonobese diabetic patients. Homocysteine metabolism that has been studied in 2 animal models of type 2 diabetes with obesity led to conflicting data. The aim of the present study was to analyze homocysteine metabolism in a spontaneous nonobese model of type 2 diabetes, the Goto–Kakizaki rats at various successive and well characterized stages of the disease: during early postnatal normoglycemia, at the onset of hyperglycemia (around weaning), and during chronic mild hyperglycemia with progressive insulin resistance. Compared to age-matched Wistar controls, Goto–Kakizaki rats showed lower plasma levels of homocysteine and a falling trend in its major byproduct antioxidant, glutathione, from the prediabetic stage onwards. Concomitantly, Goto–Kakizaki rats exhibited increased liver activity of cystathionine beta synthase, which catalyzes the condensation of homocysteine with serine in the first step of the transsulfuration pathway. These results emphasize a strong association between homocysteine metabolism and insulin via the first step of the hepatic transsulfuration pathway in Goto–Kakizaki rats.

BACKGROUND: Unlike other strains, spontaneously type 1 non-obese diabetic (NOD) mice experience transient hyperinsulinemia after weaning. The same applies for NOD/SCID mice, which lack functional lymphocytes, and unlike NOD mice, do not develop insulitis and diabetes like NOD mice. AIMS: Given that β-cell stimulation is a natural feature of gestation, we hypothesized that glucose homeostasis is disturbed in gestate pre-diabetic NOD and non-diabetic NOD/SCID mice, which may accelerate the onset of diabetes and increase diabetes prevalence. METHODS: During gestation and postpartum, mice were analyzed under basal feed conditions, and following glucose injection (1 g/kg, i.p.) after overnight fast, using glucose tolerance test (GTT). Glycemia, corticosteronemia, blood and pancreatic insulin, glucagon levels, islet size, and islet morphology were evaluated. Glycemia and mortality were assessed after successive gestations in NOD mice mated for the first time at 2 different ages. RESULTS: 1. Basal glucagonemia rose markedly in first-gestation fed NOD mice. 2. β-cell hyperactivity was present earlier in first-gestation non-diabetic fasted NOD and NOD/SCID mice than in age-matched C57BL/6 mice, assessed by increased insulin/glucose ratio after GTT. 3. Overnight fasting increased corticosteronemia rapidly and sharply in pre-diabetic gestate NOD and NOD/SCID mice. 4. Islet size increased in non-diabetic gestate NOD mice compared with C57BL/6 mice. 5. Successive gestations accelerated diabetes onset, and contributed to increased mortality in NOD mice. CONCLUSIONS: First-gestation pre-diabetic NOD and non-diabetic NOD/SCID mice exhibited β-cell hyperactivity and deregulation of glucagon and/or corticosterone secretion. This amplified normally occurring insulin resistance, further exhausted maternal β-cells, and accelerated diabetes in NOD mice.

BACKGROUND:Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra). METHODOLOGY/PRINCIPAL FINDINGS:Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia. CONCLUSIONS/SIGNIFICANCE:GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the beta-cell microenvironment and contribute to progressive type 2 diabetic beta-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent beta-cell dysfunction in type 2 diabetes.

Recent studies suggest an inflammatory process, characterized by local cytokine/chemokine production and immune cell infiltration, regulates islet dysfunction and insulin resistance in type 2 diabetes. However, the factor initiating this inflammatory response is not known. Here, we characterized tissue inflammation in the type 2 diabetic GK rat with a focus on the pancreatic islet and investigated a role for IL-1. GK rat islets, previously characterized by increased macrophage infiltration, displayed increased expression of several inflammatory markers including IL-1β. In the periphery, increased expression of IL-1β was observed primarily in the liver. Specific blockade of IL-1 activity by the IL-1 receptor antagonist (IL-1Ra) reduced the release of inflammatory cytokines/chemokines from GK islets in vitro and from mouse islets exposed to metabolic stress. Islets from mice deficient in IL-1β or MyD88 challenged with glucose and palmitate in vitro also produced significantly less IL-6 and chemokines. In vivo, treatment of GK rats with IL-1Ra decreased hyperglycemia, reduced the proinsulin/insulin ratio, and improved insulin sensitivity. In addition, islet-derived proinflammatory cytokines/chemokines (IL-1β, IL-6, TNFα, KC, MCP-1, and MIP-1α) and islet CD68 + , MHC II + , and CD53 + immune cell infiltration were reduced by IL-1Ra treatment. Treated GK rats also exhibited fewer markers of inflammation in the liver. We conclude that elevated islet IL-1β activity in the GK rat promotes cytokine and chemokine expression, leading to the recruitment of innate immune cells. Rather than being directly cytotoxic, IL-1β may drive tissue inflammation that impacts on both β cell functional mass and insulin sensitivity in type 2 diabetes.

Increasing evidence indicates that decreased functional beta-cell mass is the hallmark of type 2 diabetes (T2D) mellitus. Nowadays, the debate focuses on the possible mechanisms responsible for abnormal islet microenvironment, decreased beta-cell number, impaired beta-cell function, and their multifactorial aetiologies. This review is aimed to illustrate to what extend the Goto-Kakizaki rat, one of the best characterized animal models of spontaneous T2D, has proved be a valuable tool offering sufficient commonalities to study these aspects. We propose that the defective beta-cell mass and function in the GK model reflect the complex interactions of multiple pathogenic players: (i) several independent loci containing genes responsible for some diabetic traits (but not decreased beta-cell mass); (ii) gestational metabolic impairment inducing an epigenetic programming of the pancreas (decreased beta-cell neogenesis and/or proliferation) which is transmitted to the next generation; and (iii) loss of beta-cell differentiation due to chronic exposure to hyperglycemia/hyperlipidemia, inflammatory mediators, oxidative stress and to perturbed islet microarchitecture.

The effects of bromocriptine (10 mg/kg), known to inhibit prolactin secretion and lower autoimmune processes, were studied on glucose homeostasis in non-fasted non-obese diabetic mice, a spontaneous model of type 1 diabetes. Hyperglycemia was observed 120 and 240 min after i.p. but not s.c. injection. Bromocriptine administration i.p. led to rapid and marked hyperglycemia characterized by sexual dimorphism with males having higher glycemia than females. Bromocriptine induced a rapid but transient decrease in insulinemia in males only and biphasic increases in glucagon levels and a sustained stimulatory effect on circulating corticosterone in both sexes. Bromocriptine-induced hyperglycemia involved D2-dopaminergic receptors, as demonstrated by the inhibitory effect of the D2-dopamine antagonist, metoclopramide (10 mg/kg). Simultaneous injection of bromocriptine and metoclopramide also blocked the rise in blood corticosterone. In conclusion, by inducing hyperglycemia, i.p. bromocriptine administration to prediabetic autoimmune mice may counteract its beneficial anti-immunostimulatory effects.

Activation of the innate immune system in obesity is a risk factor for the development of type 2 diabetes. The aim of the current study was to investigate the notion that increased numbers of macrophages exist in the islets of type 2 diabetes patients and that this may be explained by a dysregulation of islet-derived inflammatory factors. Increased islet-associated immune cells were observed in human type 2 diabetic patients, high-fat–fed C57BL/6J mice, the GK rat, and the db/db mouse. When cultured islets were exposed to a type 2 diabetic milieu or when islets were isolated from high-fat–fed mice, increased islet-derived inflammatory factors were produced and released, including interleukin (IL)-6, IL-8, chemokine KC, granulocyte colony-stimulating factor, and macrophage inflammatory protein 1α. The specificity of this response was investigated by direct comparison to nonislet pancreatic tissue and β-cell lines and was not mimicked by the induction of islet cell death. Further, this inflammatory response was found to be biologically functional, as conditioned medium from human islets exposed to a type 2 diabetic milieu could induce increased migration of monocytes and neutrophils. This migration was blocked by IL-8 neutralization, and IL-8 was localized to the human pancreatic α-cell. Therefore, islet-derived inflammatory factors are regulated by a type 2 diabetic milieu and may contribute to the macrophage infiltration of pancreatic islets that we observe in type 2 diabetes.

Article suite au 41st Annual Meeting of the European-Association-for-the-Study-of-Diabetes. 10-15 September 2005, Athens, Greece; International audience; The molecular pathways leading to islet fibrosis in diabetes are unknown. Therefore, we studied gene expression in islets of 4-month-old Goto-Kakizaki (GK) and Wistar control rats. Of 71 genes found to be overexpressed in GK islets, 24% belong to extracellular matrix (ECM)/cell adhesion and 34% to inflammatory/immune response families. Based on gene data, we selected several antibodies to study fibrosis development during progression of hyperglycemia by immunohistochemistry. One-month-old GK and Wistar islets appeared to be similar. Two-month-old GK islets were strongly heterogenous in terms of ECM accumulation compared with Wistar islets. GK islet vascularization, labeled by von Willebrand factor, was altered after 1 month of mild hyperglycemia. Numerous macrophages (major histocompatibility complex class II(+) and CD68(+)) and granulocytes were found in/around GK islets. These data demonstrate that marked inflammatory reaction accompanies GK islet fibrosis and suggest that islet alterations in this nonobese model of type 2 diabetes develop in a way reminiscent of microangiopathy.

Sjogren's syndrome is an autoimmune disease in which lymphocytic infiltrates develop in the exocrine glands. Pathogenetic aspects of the disease can be studied in the nonobese diabetic (NOD) mouse strain, a spontaneous model for Sjogren's syndrome. Apoptosis may play a role in the initation phase and in the effector phase of autoimmune diseases. Here, we have examined the role of apoptosis in the development of sialoadenitis in the NOD mouse. Apoptotic cells and the expression of apoptosis-related molecules were studied in submandibular glands (SMG) of NOD and NOD-scid mice before and after the onset of sialoadenitis. Numbers of apoptotic cells were not increased as compared with control mice, at any age. By immunohistochemistry, we demonstrated increased expression of Fas, Fas ligand (FasL), and bcl-2 on SMG epithelial cells of NOD and NOD-scid mice, as early as 3 days of age. mRNA expression of Fas and FasL was also examined in SMG by RQ-PCR. Low-level expression of Fas and FasL mRNA was observed in all mouse strains, from 1 day of age onward. We conclude that increased protein expression of Fas and FasL on SMG epithelial cells of NOD and NOD-scid mice probably indicates a genetically programmed abnormality in these cells that may form a trigger for the development of sialoadenitis in NOD mice. Because increased numbers of apoptotic cells were not observed, a role for actual apoptosis in the initiation or effector phase of sialoadenitis in the NOD mouse is unlikely.

Female nonobese diabetic (NOD) mice more frequently develop autoimmune diabetes than NOD males. Orchidectomy of the latter aggravates insulitis and diabetes. Because clear differences in immune function have not been observed between prediabetic females and males, before or after castration, we hypothesized that sex-related differences in diabetes incidence are related to target organ-specific actions of sex steroids. Previously, we showed that prediabetic NOD females develop hyperinsulinemia and subsequently mega-islets. Infiltration of the first inflammatory leukocytes is predominantly associated with these mega-islets. Here, we determined the relationship between sex hormones, mega-islet formation, and infiltrating cells in NOD and nonobese diabetic/severe combined immune-deficient (NODscid) mice. Mega-islet formation was reduced in NOD males compared with NOD females, and orchidectomy increased it, indicating a relationship between androgen levels and mega-islet formation. Moreover, enhanced mega-islet formation in castrated NOD males was associated with increased numbers of infiltrating leukocytes. Castrated NODscid males also exhibited increased mega-islet formation and dendritic cell infiltration, indicating that lymphocytes are not required for castration-induced effects. In conclusion, we show that androgens influence pancreatic islets and autoimmune infiltration in NOD and NODscid mice. This suggests that the gender difference in diabetes incidence in NOD mice is related to target organ-specific androgen effects.

In the nonobese diabetic (NOD) mouse model for type 1 diabetes, the inflammatory infiltration of islets starts with an influx of dendritic cells (DC) and macrophages (Mphi) at approximately 4 weeks of age. Around this time, NOD mice show endocrine abnormalities, indicated by a transient hyperinsulinemia that lasts until 8 weeks of age. Subsequently, they develop abnormally large islets of Langerhans, here designated as "mega-islets." NODscid mice, which lack functional lymphocytes, also exhibit transient hyperinsulinemia, but to a lesser extent. First, to determine the role of lymphocytes in the morphological islet abnormalities, we compared 6-week-old (prediabetic) NOD and NODscid females regarding mega-islet development and accumulation of antigen-presenting cells (APC), particularly CD11c+ DC and ERMP23+ Mphi. In NODscid mice, early APC infiltration and mega-islets were present, but less marked compared with NOD mice, thus suggesting a role of lymphocytes in mega-islet formation. In both NOD and NODscid mice, the APC infiltration was predominantly found around the mega-islets, suggesting a relationship between both parameters. Second, to analyze the role of beta-cell hyperactivity in mega-islet formation, we studied the effect of short-term prophylactic insulin treatment on these parameters. Prophylactic insulin treatment decreased the percentages of mega-islets in both NOD and NODscid mice, indicating that beta-cell hyperactivity is also involved in mega-islet formation. In conclusion, mega-islet formation in mice with the NOD genetic background takes place under the influence of both beta-cell hyperactivity and leukocytes.

Glutamic acid decarboxylase (GAD), among other potential autoantigens, is thought to play a crucial role in type I diabetes, particularly in a spontaneous model of the disease, the nonobese diabetic (NOD) mouse. In the pancreas, the presence of GAD and gamma-aminobutyric acid (GABA), the decarboxylation product of GAD and a putative neurotransmitter in the islets of Langerhans, is well documented in the beta-cells. This is particularly true in rats, in which another GABAergic structure exists near the islets, the neuronal bodies. In this study, first the GABA content was measured in isolated islets from NOD and C57BL/6 mice (controls), and a decrease was found in NOD females as their insulitis progressed. Second, for the first time in mice, confocal analysis of immunofluorescent-labeled pancreatic sections revealed near the islets neuronal structures in which GAD and neuropeptide Y were colocalized, as they are in the brain. These structures were always observed in the pancreata of both sexes of C57BL/6 mice at the various ages investigated. In NOD mice, however, these neuronal structures were only detected in young females (10 weeks old) and in males until an intermediate age. Moreover, patches of T cells surrounding GAD-containing fibers were seen in the vicinity of the islets with incipient periinsulitis.